The present invention relates to complexes of CDK2 protein with other proteins, in particular, complexes of CDK2 with cyclin H, CDK2 with cyclin I, CDK2 with ERH, CDK2 with hsReq*-1, and CDK2 with hsReq*-2 proteins. The invention includes antibodies to CDK2 complexes, and their use in, inter alia, screening, diagnosis, prognosis and therapy. The invention further relates to the hsReq*-1 and hsReq*-2 genes and proteins and derivatives, fragments and analogs, thereof.
Human cyclin-dependent kinase 2 or cell division kinase (CDK2; GenBank Accession No. X61622; Elledge and Spottswood, 1991, EMBO J. 10: 2653-2659; Ninomiya-Tsuji et al., 1991, Proc. Natl. Acad. Sci. USA 88: 9006-9010) is a serine-threonine protein kinase of 298 amino acids that has approximately 65% amino acid identity to a second critical cell cycle regulator, p34cdc2, more commonly known as CDC2. CDK2 is expressed late in G1 or early in S phase, slightly before CDC2, and is pivotal for G1/S transition. CDK2 cannot complement yeast CDC2/CDC28 mutations under all the conditions that CDC2 can, indicating that the two kinases regulate the cell cycle at distinct stages.
CDK2 activity is dependent upon phosphorylation of threonine 160 by CDK-activating kinase (CAK), which occurs when CDK2 complexes with cyclins A and E. Conversely, CDK2 kinase activity is inactivated by dephosphorylation by human KAP (CDK-Associated Phosphatase; Poon and Hunter, 1995, Science 270: 90-93). In particular, competition between KAP and cyclin A determines the phosphorylation state of CDK2. CDK2 phosphorylates pRb, p53, transcription factor E2F, histone H1, and other proteins central to cell cycle control (Higashi et al., 1996, Eur. J. Biochem. 237: 460-467). Other proteins, including p21cip, and p27kip, complex with CDK2 to block its interaction with downstream substrates, as well as blocking CDK2 phosphorylation itself (Adams et al., 1996, Mol. Cell. Biol. 16: 6623-6633). The complex interplay of phase-specific cyclin expression, phosphorylation/dephosphorylation cascades, and other CDK2 interacting proteins such as p21waf and p27kip, ultimately plays out through CDK2 activity to determine cell cycle progression.
Deregulation of CDK2 is strongly implicated in mechanisms of carcinogenesis and in the treatment of cancer. DNA tumor viruses transform cells by directly inhibiting Rb tumor-suppressing function (e.g., papilloma viruses and cervical cancer). The Rb then releases negative control of E2F. Normally, the inhibition of Rb is accomplished by phosphorylation of Rb by CDK2 (Nevins, 1992, Science 258: 424-429). CDK2 is implicated in the differentiation of glioma cells (Kokunai et al., 1997, J. Neuro-oncol. 32: 125-133). In human breast carcinoma cells, the anti-cancer agent flavopiridol induces G1 arrest by inhibition of both CDK2 and CDK4 (Carlson et al., 1996, Cancer Res. 56: 2973-2978). Anti-estrogens up-regulate CDK2 inhibitors p21cip and p27kip, thus causing reduction in pRb phosphorylation, and decreased cell progression into S phase (Watts et al., 1995, Mol. Endocrinol. 9: 1804-1813). Serum-deprivation of vascular smooth muscle cells is associated with CDK2/p27kip complex formation, leading to inhibition of CDK2 enzymatic activity (Chen et al., 1997, J. Clin. Invest. 99: 2334-2341). Thus, along with regulation of cyclin A expression, CDK2 activity is the mechanism through which p27kip acts to inhibit intimal hyperplasia during atherosclerosis and re-stenosis.
To review, CDK2 is implicated in the control of cell cycle progression, transcriptional regulation via E2F, control of cellular differentiation, intracellular signal transduction involving phosphorylation, mechanisms of tumorigenesis, tumor progression and spread, and athero-sclerosis and re-stenosis via effects on intimal proliferation.
Human cyclin H (GenBank Accession No. U11791; Makela et al., 1994, Nature 371: 254-257) is a 323 amino acid protein that complexes with CDK7 to form CDK-activating kinase (CAK; Fisher and Morgan, 1994, Cell 78: 713-724). CAK, in turn, phosphorylates CDK2, as well as various cyclin complexes of CDK2 and CDK4. Thus, like CDK2, cyclin H is centrally implicated in control of cell cycle progression. CAK is also associated with the mammalian transcription factor IIH (TFIIH), a multisubunit complex that is required for transcription and DNA nucleotide excision repair (Drapkin et al., 1996, Proc. Natl. Acad. Sci. USA 93: 6488-6493). Therefore, the role of cyclin H extends beyond cell cycle control to include coordination of the cell cycle with transcription and DNA repair. Dysfunction of TFIIH is implicated in various genetic disorders including xeroderma pigmentosum, Cockayne""s syndrome and trichothiodystrophy (Seroz et al., 1995, Curr. Opin. Genet. Dev. 5: 217-222). In summary, cyclin H is implicated in the control of cell cycle progression, transcriptional control via TFIIH, DNA repair, and various genetic disorders associated with impaired DNA repair.
Cyclin I (GenBank Accession No. D50310; Nakamura et al., 1995, Exp. Cell Res. 221: 534-542), in contrast to other cyclin proteins, is widely expressed in many post-mitotic tissues at constant levels throughout the cell cycle. The protein contains a typical cyclin box near the N-terminus, implicating it in control of cell cycle progression and transcriptional control (Gibson et al., 1994, Nucleic Acids Res. 22: 946-952). It also has a PEST domain proximal to its C-terminus; thus, it may be the target of rapid inactivation via ubiquitin-based proteolysis, as are most transcription factors (Rechsteiner, 1990, Semin. Cell Biol. 1: 433-440).
A human cDNA (GenBank Accession No. D85785; Isomura et al., 1996) encoding a 104 amino acid protein termed ERH, homologous to DROER, the enhancer of the rudimentary gene in Drosophila melanogaster, was found to interact with CDK2. In Drosophila, the gene product is required for transcriptional regulation of the rudimentary gene. The enzyme functions in the pyrimidine metabolic pathway, and has a critical role in wing development. ERH is thus implicated in transcriptional control, DNA pyrimidine metabolism, and in development.
Two sequences were identified as CDK2 interactants which are identical to sequences within the human homolog of the mouse zinc finger protein Requiem (hsReq; GenBank Accession No. U94585; Gabig et al., 1994, J. Biol. Chem. 269: 29515-29519). HsReq is hypothesized to encode a transcription factor required for apoptosis following survival factor withdrawal from myeloid cells and to function as a tumor suppressor. However, as described infra, these hsReq regions identified as encoding a protein that interacts with CDK2 must occur with a splice variant of hsReq containing amino acids encoded by a nucleotide sequence of the 3xe2x80x2 untranslated region of the hsReq mRNA, which was identified as encoding a CDK2 interacting protein. Two such splice variants are described in Section 5.2, and are referred to as hsReq*-1 and hsReq*-2.
CDK2 complexes with any of cyclin H, cyclin I, ERH, hsReq*-1 or hsReq*-2 have not been previously described.
Citation of a reference herein shall not be construed as an admission that such is prior art to the present invention.
The present invention provides compositions and methods of production of protein complexes of CDK2 with proteins that interact with (i.e. bind to) CDK2 (the proteins shown to bind with CDK2 are designated xe2x80x9cCDK2-IPxe2x80x9d for CDK2 interacting protein, and a complex of CDK2 and a CDK2-IP is designated as CDK2:CDK2-IP herein). Specifically, the invention relates to complexes of CDK2, and derivatives, fragments and analogs of CDK2 with cyclin H. with cyclin I, with ERH, with hsReq*-1 and with hsReq*-2, and their derivatives, analogs and fragments.
In their screen for proteins that interact with CDK2, the present inventors have identified novel proteins, hsReq*-1 and hsReq*-2, which are encoded by mRNA splice variants of the hsReq gene, i.e., the mRNAs encoding hsReq*-1 and hsReq*-2 are generated by RNA splicing at splice sites other than the splice sites used to process the mRNA encoding hsReq. Accordingly, the invention further relates to nucleotide sequences of hsReq*-1 and hsReq*-2 (human hsReq*-1 and hsReq*-2 genes and homologs of other species), as well as derivatives (e.g., fragments) and analogs thereof. Nucleic acids able to hybridize to or complementary to the foregoing nucleotide sequence, such as the inverse complement (i.e., has the complementary sequence running in reverse orientation to the strand so that the inverse complement would hybridize without mismatches to the nucleic acid strand; thus, for example, where the coding strand is hybridizable to a nucleic acid sequence with no mismatches between the coding strand and the hybridizable strand, then the inverse complement of the hybridizable strand is identical to the coding strand) of the foregoing sequences are provided. In particular, the invention provides nucleic acids that comprise, as well as nucleic acids (e.g., the inverse complement) that are hybridizable to or complementary to, at least a 5, 10, or 25 nucleotide portion of the nucleotide sequences encoding hsReq*-1 and hsReq*-2 that span the alternate splice junctions of the hsReq*-1 and hsReq*-2 mRNAs (i.e. the point in the hsReq*-1 or hsReq*-2 nucleotide sequence at which the 5xe2x80x2 and 3xe2x80x2 splice sites were joined in processing the hsReq*-1 or hsReq*-2 mRNA). The invention also relates to hsReq*-1 and hsReq*-2 derivatives and analogs that are functionally active, i.e., they are capable of displaying one or more known functional activities of a wild-type hsReq*-1 or hsReq*-2 protein. Such functional activities include, but are not limited to the ability to bind with [or compete for binding with] CDK2, antigenicity [ability to bind (or compete with hsReq*-1 or hsReq*-2 for binding) to an anti-hsReq*-1 or anti-hsReq*-2 antibody, respectively], and immunogenicity (ability to generate an antibody that binds hsReq*-1 or hsReq*-2, respectively). Specific embodiments relate to fragments, or derivatives or analogs thereof, comprising all or a portion of amino acids 187 to 280 of the hsReq*-1 amino acid sequence as depicted in FIG. 6 (SEQ ID NO:11) and comprising all or a portion of amino acids 188 to 210 of the hsReq*-2 amino acid sequence as depicted in FIG. 7 (SEQ ID NO:13).
Methods of production of the CDK2:CDK2-IP complexes and of hsReq*-1 and hsReq*-2 proteins, and derivatives and analogs of the complexes and proteins, e.g., by recombinant means, are also provided. Pharmaceutical compositions are also provided.
The invention further provides methods of modulating (i.e., inhibiting or enhancing) the activity of CDK2:CDK2-IP complexes, particularly CDK2:cyclin H, CDK2:cyclin I, CDK2:ERH, CDK2:hsReq*-1 or CDK2:hsReq*-2 complexes. The protein components of the complexes have been implicated in cellular functions, including but not limited to: control of cell cycle progression, cellular differentiation and apoptosis, hyperproliferative disorders including tumorigenesis and tumor progression; degenerative disorders; regulation of transcription; control of intracellular signal transduction involving phosphorylation; intimal hyperplasia, re-stenois, atherosclerosis, and neovascularization; pyrimidine metabolism, and various genetic disorders associated with impaired DNA repair.
Accordingly, the invention provides methods of screening CDK2:CDK2-IP complexes, particularly complexes of CDK2 with cyclin H, cyclin I, ERH, hsReq*-1 or hsReq*-2, and the hsReq*-1 and hsReq*-2 proteins, as well as derivatives and analogs of the CDK2:CDK2-IP complexes and hsReq*-1 and hsReq*-2 proteins for the ability to alter cell functions, particularly those cell functions in which CDK2 and/or a CDK2-IP has been implicated, such as but not limited to tumorigenesis and other hyperproliferative disorders, arthritis and neurodegeneration, atherosclerosis and other cardiovascular diseases, and various genetic disorders.
The present invention also relates to therapeutic and prophylactic as well as diagnostic, prognostic, and screening methods and compositions based upon CDK2:CDK2-IP complexes (and the nucleic acids encoding the individual proteins that participate in the complexes) as well as hsReq*-1 and hsReq*-2 proteins and nucleic acids. Therapeutic compounds of the invention include, but are not limited to, CDK2:CDK2-IP complexes and complexes where one or both members of the complex is a derivative or analog of CDK2 or a CDK2-IP; hsReq*-1 and hsReq*-2 proteins and derivatives, fragments and analogs thereof; antibodies to and nucleic acids encoding the foregoing; and antisense nucleic acids to the nucleotide sequences encoding the complex components and hsReq*-1 and hsReq*-2 antisense nucleic acids. Diagnostic, prognostic and screening kits are also provided.
Animal models and methods of screening for modulators (i.e. agonists, antagonists and inhibitors) of the activity of CDK2:CDK2-IP complexes and hsReq*-1 and hsReq*-2 proteins are also provided.
Methods of identifying molecules that inhibit, or alternatively, that increase formation of CDK2:CDK2-IP complexes are also provided.